The Effects And Mechanisms Of Ang-(1-7) And Ang? On Blm-induced Lung Fibrosis In Rats

BACKGROUPPulmonary fibrosis, which has great influence on the health of millions of people around the world, is a kind of interstitial disease with progressive collagen deposition,the pathological and physiological characteristics of which are alveolar injury, pulmonary interstitial inflammatory cell infiltration, fibroblast's activation and proliferation and deposition of extracellular matrix,leading to damage of lung structure or even severe dyspnea. It is considered to be the key link in the initiation and development of pulmonary fibrosis that the activation and proliferation of fibroblast may result in deposition of extracellular matrix, generation of cytokines and regulation functions of the type II epithelial and any other cells.Angiotensin? (Ang?), is proven to promote the accumulation of inflammatory cells and the secretion of inflammatory factors from inflammatory cells.Besides, Ang? is an important factor to enhanced the activity of fiibroblasts,which can express more DNA to promote proliferation and secrete extracellular matrix. Recent reports have demonstrated local renin-angiotensin system(RAS) in lung tissue was excessive activation, Ang? was over expression and Angiotensin? type 1 receptor(AT1R) had a improved expresss.On one hand, Excessive activation of the RAS may damage alveolar epithelial cells, promote alveolar epithelial cell apoptosis, induce fibroblast proliferation and migration. One the other hand,RAS may promote the development of pulmonary fibrosis by the way of directly activating fibroblasts, promoting cell's proliferation and differentiation and secreting more extracellular matrix.Thus,down-regulation or blockade of RAS become an effective approach of curing pumonary fibrosis.In animal experiments,using angiotensin converting enzyme inhibitors (ACE2) and angiotensin-converting enzyme inhibitor, ACEI) may attenuate pulmonary fibrosis induced by bleomycin.in addition,ARB may improve the lung function in Clinical group though the effect of reduction of lung fibrosis is not so satisfaction.Therefor,it may not have a great improvement in pulmonary fibrosis until we can find a druf to down-regulate or block RAS.Angiotensin-converting enzyme 2 (ACE2)/Angiotensin (1-7) (Ang-(1-7)/ Mas axis, which is a very important components of RAS, counteracts the effect of ACE/Ang?/ATIR. Ang-(1-7) is producted by ACE2 hydrolyzing Ang? which can against Ang? effect, for instance, the anti-proliferation, regulating water and electrolyte balance, diastolicing blood vessels, lower blood pressure, and so on. It has been proved that Over-expression of ACE2 and Ang-(1-7) in rats can inhibit heart remodeling, liver fibrosis, kidney fibrosis and protect brain function, reduce BLM-induced rat lung fibrosis. But, we do not understand the mechanism about how reduce BLM-induced rat lung fibrosis and whether there are some connections with inhibition of Ang-(1-7) to Ang??Futhur studies shown that Ang? could promote the phosphorylation of ERK via activating AT1R which is special receptor of Ang?. Phosphor-ERK activate inhibitor kappa B kinase (I?K) and then active I?K induces NF-?B activation through the phosphorylation and degradation of I?B. The liberated NF-?B translocates to the nucleus, where it binds to specific promoters and regulates target gene expression, including transforming growth factor (TGF-pl) and connective tissue growth factor (CTGF). Over-expression of profibrotic factors could activate fibroblasts, induce differentiation and the accumulation of collagen.Then, whether Ang-(1-7) could suppresse lung fibrosis via inhibiting ERK/NF-?B activated by Ang??METHODIn vivo, To determine whether Ang-(1-7) exerts anti-fibrotic effect on BLM or Ang? induced pulmonary injury, meanwhile Ang-(1-7) itself initiated pro-inflammatory response, we established two kinds of animal models as following:(1) Twenty-four wistar male rats were randomly divided into four groups, each containing 6 rats, including control group, BLM treatment group, BLM+Ang-(1-7) treatment group and BLM+Ang? treatment group. Rats under the influence of pentobarbital anesthesia received a single intratracheal instillation of 5 mg/kg dose of BLM sulfate in 200?l of sterile saline. Meanwhile, micro-osmotic pumps were implanted subcutaneously allowing 28 days of continuous infusion with Ang?or Ang-(1-7) at a rate of 25?g/kg1·h-1. Control rats received intratracheal instillation of 200?l of sterile saline and chronic infusion with saline subcutaneously. (2) In the subsequent study, twenty-four wistar male rats were randomly divided into four groups, including control group, Ang-(1-7) treatment group, Ang? treatment group, Ang?+Ang-(1-7) treatment group. Micro-osmotic pumps were implanted subcutaneously to the rats allowing 28 days of infusion with Ang? or (and) Ang-(1-7) at a rate of 25?g/kg-1·h-1. Chronic infusion with saline subcutaneously acted as control. Histological changes of the lungs were evaluated by haematoxylin and eosin (H&E) stain and Masson's trichrome stain. Lung relevant protein level was assessed by immunohistochemistry and western blotting.In vitro, lung fibroblast cultured from lung of rat. Cells were cultured in a-DMEM (GIBCO BRL, USA) with antibiotics and 15% fetal bovine serum. Cells were passaged every 3-5 days in 60-mm dishes. HFL-1 cells were grown in 5% CO2 at 37? in a humidified atmosphere. Experiments were performed on cells at passages 3-5. After reaching near confluence, the cells were rested for 24 hours in serum-free medium. Cells were preincubated respectively for 1 hour with irbesartan (10-5M), A-779 (10-5M), PD98059 (10-5M) and BAY117082 (10-5M) before exposure to Ang? (10-7M) or Ang-(1-7) (10-7M) for indicated time. The mRNA expressions of TNF-a and PAI-1 were detected by qRT-PCR. Western blot were used to determine the phosphorylation of ERK and I?K and protein expression of I?B?NF-?B? CTGF??-SMA and a-collagen I.RESULT In vivo1. Ang-(1-7) reduced BLM-induced lung fibrosis in rats.The HE stain of sections from rat lung tissue shown:the lung tissue of control group was very normal by microscopic observation and unfound abundant of inflammative cell and accumulation of collagen in pulmonary interstitium. Microscopically, there were a lot of alveolar structure damage, obviously thicken of alveolar interval, inflammatory cells infiltration and fibroblast proliferation in great quantities, fibrous tissue scarring change assumes the strip samples, patchy distribution, but peripheral inflammation of the lung parenchyma a better absorption. It is less alveolar structure damage, infiltration of inflammatory cells in alveolar interval, proliferation of fibroblast and deposition of extracellular matrix than BLM group in BLM+Ang-(1-7) group. BLM treatment resulted in a higher Ashcroft score of pulmonary fibrosis than control, but chronic infusion with Ang-(1-7) resulted in a significantly lower Ashcroft score. By Massion trichromatic dyeing, we observed that there was a small amount of collagen fibers dyed blue in alveolar interval of control group rat. Alveolar interval of dyed blue collagen fibers was very abundant in BLM group, and Ang-(1-7) treatment group significantly decreased alveolar interval dyed blue collagen fibers than BLM group. Lung hydroxyproline (HYP) content of control group was 0.5952±0.5952 mg/g; BLM group was 3.6905±3.6905 mg/g; BLM+Ang-(1-7) group significantly reduced from 2.1429± 2944 mg/g than BLM group.Pro-Fibrotic factors, including TNF-?, CTGF and collagen degradation enzyme PAI-1, were detection that shown BLM significantly increased the expression of them than control group, while Ang-(1-7) treatment group was reduction compared with BLM group. The expression of ?-SMA and collagen proteins of extracellular matrix ingredient increased significantly in BLM group than control group, while Ang-(1-7) treatment group was reductive compared with BLM group. Ang? aggravated BLM induced pulmonary fibrosis. 2. Ang-(1-7) decreased phosphor-ERK and activation of NF-?B of lung tissue in BLM-treated ratsThe ratio of p-ERK/ERK was significantly increased in BLM group and Ang? aggravated this ratio, but Ang-(1-7) reduced the phosphorylation of ERK. In cytoplasm protein, p-I?K was significantly increased in lung fibrosis models and Ang? did not elevate the the phosphorylation of I?K on the base of BLM, Ang-(1-7) cut down the ratio of p-I?K/I?K. On another hand, BLM induced the hydrolysis of IkB and Ang-(1-7) blocked that. The expression of NF-?B protein was up-regulated in nuclear protein of BLM group and Ang? accelerated the level, however, Ang-(1-7) take a reverse role compare with BLM about The expression of NF-?B protein in nuclear protein.3. Ang-(1-7) suppressed ACE/Ang?/AT1R axis, but activated ACE2/Ang-(1-7)/Mas axis.To test the activation of local RAS, ACE protein and AT1R mRNA increased significantly compared with normal group in BLM group. ACE2 protein and Mas mRNA level were only slightly increased in BLM group than control group. The expression levels of ACE protein and AT1R mRNA were significantly decreased compared with BLM group in BLM+Ang-(1-7) group. However, ACE2 protein and Mas mRNA expression level increased significantly. In vitro1. Ang-(1-7) inhibited the activation of ERK/NF-?B pathway induced by Ang?.The phosphorylation of ERK was increased after Ang? stimulating 3omin by western blot and Ang-(1-7) also induced the phosphorylation of ERK, but the effect was weaker than Ang?. However, Ang-(1-7), irbesartan and PD98059 reduced the phosphorylation of ERK after Ang? stimulating. A-779 abolished the effect of Ang-(1-7) to Ang?. Ang?and Ang-(1-7) induced the nuclear translation of NF-kB by Immunofluorescence, which were inhibited by Ang-(1-7) and irbesartan. We further detected the change of nuclear protein NF-?B and cytoplasm protein I?K?/? and I?B-?:and found Ang? promoted expression of nuclear protein NF-?B and the phosphorylation of I?K?/?, but inhibit the expression of I?B-?. Ang-(1-7) had a similar effect like Ang?, but slightly. Ang-(1-7)?irbesartan?PD98059 and BAY117082 suppressed the effect of Ang? in stimulating NF-?B pathway. The inhibitive effect of Ang-(1-7) to Ang?was reduced by A-779. Ang?, Ang-(1-7), irbesartan, PD98059, BAY117082 and A-779 play similar effect in the effect of TNF-? mRNA and CTGF protein like in NF-?B pathway.2. Ang-(1-7) reduced the expression of a-SMA and a-collagen I induced by Ang? in fibroblast.Ang? promoted expression of a-SMA and a-collagen and Ang-(1-7) had a similar effect like Ang?, but slightly. Ang-(1-7)?irbesartan?PD98059 and BAY117082 suppressed the effect of Ang? in stimulating expression of a-SMA and a-collagen. The inhibitive effect of Ang-(1-7) to Ang? was reduced by A-779.CONCLUTION1. Ang-(1-7) reduced the BLM-induced lung fibrosis in rats. Its mechanism may be suppressing ACE/Ang?/AT1R axis and inhibiting Ang?, up-regulating ACE2/Ang-(1-7)/Mas axis.2. Ang-(1-7) suppressed the activation of ERK/NF-?B stimulated by Ang? via Mas receptor, resulting in the decrease of ECM.3. Ang-(1-7) played dual roles in fibroblast. Ang-(1-7) along promoted the synthesis and secretion of extracellular matrix in fibroblast; on the other hand, Ang-(1-7) inhibited the effect of Ang? on fibroblast.